Flow-controlled magnetic particle manipulation

ABSTRACT

Inventive methods and apparatus are useful for collecting magnetic materials in one or more magnetic fields and resuspending the particles into a dispersion medium, and optionally repeating collection/resuspension one or more times in the same or a different medium, by controlling the direction and rate of fluid flow through a fluid flow path. The methods provide for contacting derivatized particles with test samples and reagents, removal of excess reagent, washing of magnetic material, and resuspension for analysis, among other uses. The methods are applicable to a wide variety of chemical and biological materials that are susceptible to magnetic labeling, including, for example, cells, viruses, oligonucleotides, proteins, hormones, receptor-ligand complexes, environmental contaminants and the like.

[0001] This invention was made with Government support under ContractNumber DE-AC 0676RL01830 awarded by the U.S. Department of Energy. TheGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to systems for the magneticcollection of magnetically-responsive particles (referred to herein as“magnetic particles”), and the subsequent controlled dispersion of theparticles. In particular, the invention relates to the detection,isolation, separation and/or manipulation of target substances, such asfor example, chemicals and/or biological substances such asbiochemicals, cells, cell components, bacteria, viruses, toxins, nucleicacids, hormones, proteins, receptor-ligand complexes, other complexmolecules, or combinations thereof, by selective interaction thereofwith magnetic particles, which can be separated from a medium, andsubsequently resuspended for further analysis, isolation or other use.

[0003] As a background to the invention, many techniques are known andused in the prior art that involve identification, separation, and/ormanipulation of target entities, such as cells or microbes, within afluid medium such as bodily fluids, culture fluids or samples from theenvironment. It is readily appreciated that such techniques oftenrequire multiple washing and binding steps. These steps are conducted inmany such techniques by tedious manual pipetting and decantingprocedures or by using automated robotic systems that are large and notfield-portable. Such identification, separation and/or manipulationtechniques may involve, for example, analyzing a sample to determinewhether a specific pollutant, toxin or other substance is present and,if so, in what quantity; extracting a specific target substance, such asnucleic acid fragments, proteins, enzymes or the like from a sample forsubsequent processing or utilization; or the like. It is readilyappreciated that such techniques often must exhibit significantsensitivity, especially when the target substance is present in tracequantities, in order to provide accurate and useful information or toprovide a suitable specimen in an isolation technique.

[0004] One example of a technique that involves separating, isolating orotherwise manipulating organic molecules in trace is an immunoassaytechnique using antibodies as the analytical reagents. The principle ofimmunoassays is well understood. Antibodies recognize a specific analyteby following specific interactions. For low molecular weight analytessuch as drugs or metabolites, it is customary to perform competitiveimmunoassays. Typically, a fixed, limited quantity of specific antibodyis allowed to incubate with a known concentration of labeled analyte anda sample possibly containing some unknown concentration of that analyte.The quantity of label bound to antibody is inversely proportional to theamount of analyte in the test specimen.

[0005] Although there is a high number of immunoassay techniques, only afew are widely used. Among these are indirect techniques where theanalyte is measured through a label species conjugated with one of theimmunoreagents. For quantitation, it is customary to perform abound/free separation so that labeled analyte associated with theantibody can be detected. If one of the components of the immunologicreaction is immobilized on a solid phase (heterogeneous assay), theexperimental procedure is simplified. A heterogeneous immunoassay thatinvolves competitive binding of the analyte and an enzyme-labeledanalyte to the antibody is called an Enzyme Linked Immunosorbent Assay(ELISA).

[0006] For analytes which have at least two distinguishable antigenicdeterminants, a simpler and more precise approach is to perform asandwich immunoassay, which uses a first antibody directed to oneantigenic site as a capture antibody and a second antibody directed atanother characteristic determinant as the signal generating antibody.Thus, if the capture antibody is separated from solution, or bound onsome solid support, the only way in which signal antibody can be boundto solid support or separated from solution is via analyte. Theadvantages of sandwich assay are that: (1) signal is directlyproportional to analyte concentration on the low end of the analytecurve; (2) extreme sensitivity can be obtained on the low concentrationend; (3) sandwich assays are assays of “excesses” since capture antibodyand label antibody are typically in excess of analyte and so error ismainly related to accuracy of sample input; and (4) a wide dynamicanalyte detection range (as much as 4-5 logs) is possible. Sandwichassay technology, like competitive assay, employ a wide range of systemsfor performing bound/free separations. Typically in such assays,antibodies for the analytes of interest are placed with great precisionon a solid support so as to permit analyte binding to take place on thebound antibodies. Next, solution is added which causes unbound andnon-specifically bound analyte to be carried from the binding region.Then a second labeled antibody is added, and solution is added to washaway the unbound secondary antibody. If the secondary antibody is enzymelabeled, then enzyme substrates are added to result in a detectablesignal (e.g. color change, fluorescence, conductivity change) which willbe proportional to the quantity of enzyme (and therefore analyte)specifically bound.

[0007] There are numerous ways for performing the bound/free separationutilizing a specific binding substance immobilized on a solid phase,such as, for example, antibody adsorbed or covalently linked to theinside of a tube (coated tube assay), or, more recently, affixed to amobile solid phase, such as, for example, elongate structures that canbe submerged in a liquid and then withdrawn, or beads, which can eitherbe centrifuged or separated with filters or magnetically. Typically, aseparation system should have the characteristics that the separationcan easily be performed, excess reagent can be removed easily andnon-specifically bound analyte can be washed free of the immobilizedantibody with its specifically bound, labeled analyte.

[0008] Detection of signal or radiant-energy response in an assay suchas those discussed above may be accomplished through a variety oftechniques. One example is fluorescent detection of a fluorescentlylabeled antibody, analyte or other small molecule that could beassociated with the analyte. Radioactive detection is also a possibilityif system components are impervious to the type of radioactive emissiondetected. Colorimetric detection of a dye attached to the antibody oranalyte, possibly enclosed in a liposome, is also possible.Chemiluminescent, bioluminescent, electrochemiluminescent, or enzymaticdetection is also possible, provided the substrate for the detectionreaction become available after the bound/free separation. Thus, avariety of methods exist to obtain a readable signal or otherradiant-energy response.

[0009] One major challenge in the use of immobilized binding substancesis the limited lifetime of a chemically selective surface, especiallythose that include biomolecules (often referred to as “biosensors”).Many biomolecules in aqueous solution at room temperature willchemically degrade over time and typically have a lifetime ranging fromonly hours up to about 1 week. Therefore, it is not feasible to keep abiosensor surface immersed in aqueous solution continuously for longperiods of use. The solution composition also effects the lifetime ofbiosensors. The structure and function of biomolecules and biologicalmaterials are sensitive to environmental conditions such as saltconcentration, pH and temperature. Changes in solution composition andtemperature can irreversibly denature proteins so that they will nolonger bind to specific ligands. The use of whole cells in sensorsystems is also challenging since cells require the correct mix ofmetabolites to remain viable, and solution composition and solution flowrate effect the growth rate of viable cells. The specificity andtherefore irreversible nature of many biospecific interactions alsocontributes to the limited lifetime of biosensing surfaces. Selectiveinteractions such as antibody-antigen interactions are essentiallyirreversible over the time course of minutes. Harsh reagents may be usedto remove bound antigens (and non-specifically bound molecules);however, this decreases the subsequent binding activity of the antibodyitself so that the regenerated sensing surface is not as effective asthe fresh sensing surface, negatively impacting assay accuracy andreliability. In addition, the lifetime of a biosensor is often alsolimited due to “fouling” of the sensor surface caused by thenon-specific binding of materials in a sample matrix onto the biosensorsurface.

[0010] These problems have resulted in increased use of renewablesurfaces for biosensing in which the chemically selective chemistry ison the surface of small particles. Fresh aliquots of derivatizedparticles can therefore be used for each analysis. After such ananalysis, the derivatized particles can be flushed from the system andnew particles are used for a subsequent analysis.

[0011] Where the derivatized particles are magnetic particles, isolationof the particles from media during analysis can be achieved usingmagnetic separation or high gradient magnetic separation (HGMS). As usedherein, the term “magnetic” is intended to refer to a property of aparticle whereby a force is exerted thereon by the application of amagnetic field thereto. In magnetic separation, particles of relativelylarger size (i.e., about 0.5 microns or greater in diameter) arecaptured or separated and in HGMS, smaller particles, such as, forexample, colloidal magnetic particles are separated.

[0012] Over the past several years, sub-millimeter-scale, automatedflow-based analyzers and chemical detector arrays have steadilyapproached the technology level needed for commercialization.Development is continuing toward ever more compact diagnostic analyzersfor automated immunoassays, DNA purification and amplification, cellseparation, environmental contaminant detection and the like.

[0013] In light of this background, there remain needs for furtherdevelopment of systems for the magnetic collection of magneticparticles, and the subsequent controlled resuspension of the particles.In particular, a need exists for further development of systems for thespecific detection, isolation, separation and/or manipulation of targetsubstances by selective interaction thereof with magnetic particles,which can be separated from a medium, and subsequently resuspended forfurther analysis, isolation or other use. The present inventionaddresses these needs.

SUMMARY OF THE INVENTION

[0014] The present invention provides novel systems for conductingmagnetic particle separations and resuspensions in one or more fluidmedia. In accordance with one aspect of the invention, magnetic particlecapture and release are controlled by controlling the rate of fluid flowthrough a capture zone that passes through a fixed magnetic field. Atlow flow rates through the capture zone, the particles can be capturedreproducibly and quantitatively. A high flow rate through the capturezone displaces the particles from the magnetic field.

[0015] The invention provides systems that facilitate isolation oftarget substances by providing methods for manipulating magneticparticles both during and after the binding of target substances ontothe magnetic particles. The target substance can then be tested, ifdesired, using one or more detection or analysis systems, such asluminescence detectors, spectrophotometric analysis, fluorometers, massspectrometers, flow cytometers, hematology analyzers, or other cellcounting or analytical devices.

[0016] The term “target substance” as used herein, refers to a widevariety of substances of biological, medical or environmental interestwhich are measurable individually or as a group. Examples include cells,both eukaryotic (e.g., leukocytes, erythrocytes or fungi) andprokaryotic (e.g., bacteria, protozoa or mycoplasma), viruses, cellcomponents, molecules (e.g., proteins), macromolecules (e.g., nucleicacids-RNA, DNA), and chemicals (e.g., pesticides, herbicides,explosives). Cell-associated target substances include, for example,components of the cell membrane, cytoplasm or nucleus. Among suchcell-associated structures are membrane-bound proteins or glycoproteins,including cell surface antigens of either host cell or viral origin,histocompatibility antigens, or membrane receptors. These targetsubstances may be bound as discrete entities or in the form of complexesor aggregates. Such separations are accomplished using methods of theinvention which rely on the interaction of the binding substance with atleast one characteristic target substance or analyte of interest.

[0017] Binding of the target substances to the magnetic particles iscontrolled by the surface chemistry of the magnetic particles. Thechemistry of the magnetic particles may be used for binding targetsubstances via non-specific interactions, such as, for example,electrostatic interactions, van der Waals interactions, dipole-dipoleinteractions and/or hydrogen bonding interactions. For example, where itis desired to isolate all or substantially all positively chargedproteins in a sample, this can be accomplished in accordance with theinvention using negatively charged magnetic beads. Similarly, positivelycharged magnetic beads can be used to bind all or substantially all DNA(which is negatively charged) in a sample. Alternatively, the magneticparticles may be chemically modified to include specific bindingsubstances that interact selectively with the target substances.Representative examples of binding substances that can be used inaccordance with the invention include antigens, antibodies, proteinreceptors, ligands, oligonucleotides, streptavidin, avidin, biotin andlectin. One class of specific binding substance that is used toselectively interact with the target substance is the class ofantibodies capable of immunospecifically recognizing antigens. The term“antibody” as used herein includes immunoglobulins, monoclonal orpolyclonal and immunoreactive immunoglobulin fragments. Thus, examplesof characteristic target substances and their specific bindingsubstances are: receptor-hormone, receptor-ligand, agonist-antagonist,RNA or DNA oligomers-complimentary sequences, Fc receptor of mouseIgG-protein A, avidin-biotin and virus-receptor. Still other specificbinding pair combinations that may be determined using the methods ofthis invention will be apparent to those skilled in the art.

[0018] When used for immunoassay, the instant invention provides ahighly sensitive, but relatively small-scale system for the collectionof analyte. The speed and reproducibility of collection and resuspensionare important features for automating all of the binding, washing, andmixing steps needed using small volumes to achieve a relatively low-costtest for analytes. When used for performing an immunoassay or other suchanalysis, the instant invention makes possible a significant reductionin volume for all types of samples, resulting in higher concentrationsof the target entity, thus permitting shorter analysis time. This isachievable without the need for removing, reversing or minimizing themagnetic field used to capture the magnetic particles

[0019] As such, according to one aspect of this invention, a method isprovided for separating magnetic particles from a non-magnetic carriermedium containing the particles and then resuspending the separatedmagnetic particles in a suitable dispersion medium. The carrier mediumand the dispersion medium can be the same or different. The methodinitially involves providing a fluid flow path that extends through amagnetic field source, and introducing the carrier medium/magneticparticle mixture into the fluid flow path. A magnetic field interceptsthe fluid flow path and captures magnetic particles in the fluid flowpath when the mixture flows through the capture zone at a predeterminedcapture rate. The magnetic particles are released only when the same ora different medium is caused to flow through the capture zone at ahigher rate effective to move the particles from the capture zone.

[0020] A method in accordance with the invention includes: (1) providinga fluid flow path having first and second ends and a fluid flowcontroller effective to variably impose a positive or negative pressureon the flow path to cause controlled fluid flow through the fluid flowpath in a first or second direction at predetermined rates, and acapture zone between the first and second ends, wherein a fixed magneticfield intercepts the fluid flow path in the capture zone; (2) providingin the fluid flow path a first mixture including a plurality of solidmagnetic particles dispersed in a carrier medium; (3) passing the firstmixture through the capture zone at a first predetermined capture ratewhereby a major portion of the magnetic particles become trapped in thecapture zone by the force of the magnetic field and thereby separatedfrom the carrier medium to form a first magnetic particle isolate; (4)perfusing the first magnetic particle isolate with a first dispersionmedium; and (5) pulsing the first dispersion medium through the capturezone at a first predetermined dispersion rate effective to dislodge thefirst magnetic particle isolate from the capture zone, move the magneticparticles from the magnetic field, and suspend the magnetic particles inthe first dispersion medium to provide a second mixture. If desired, themagnetic particles can be captured and released multiple times. Thisprocedure can be used to enhance mixing and therefore molecular captureefficiency from a small fluid volume. In addition, the same fluid can beused as the carrier medium and the dispersion medium. Thus, the firstand second mixtures can be the same. The capture and release can occurwithin the same volume of fluid by reversing the fluid flow directionthrough the capture zone during the capture and release functions.Alternatively, the capture and release can be into fresh volumes offluid that are moved through the capture zone.

[0021] According to another aspect of the present invention, there isprovided an apparatus for separating magnetic particles from anon-magnetic fluid medium containing such particles. The apparatusincludes: (1) a fluid flow path, the fluid flow path having first andsecond ends and a capture zone between the first and second ends; (2) afluid flow controller effective to variably impose a positive ornegative pressure on the flow path to cause controlled fluid flowthrough the fluid flow path in a first or second direction atpredetermined rates; (3) a magnetic field source generating a fixedmagnetic field, the source positioned in a fixed relationship to thefluid flow path whereby the field intercepts the fluid flow path in thecapture zone; and (4) a detector positioned to detect a physical orchemical property of a fluid in the flow path.

[0022] It is an object of the invention to provide novel systems andmethods for capturing and resuspending magnetic particles that providealternatives to systems and methods of the prior art.

[0023] Further forms, embodiments, objects, advantages, benefits,aspects and features of the present invention will be apparent from thedrawings and detailed description herein.

BRIEF DESCRIPTION OF THE FIGURES

[0024] Although the characteristic features of this invention will beparticularly pointed out in the claims, the invention itself, and themanner in which it may be made and used, may be better understood byreferring to the following descriptions taken in connection with theaccompanying figures forming a part hereof.

[0025]FIG. 1 is a schematic view of a first embodiment of a system inaccordance with the invention.

[0026]FIG. 2 is a schematic view of a second embodiment of a system inaccordance with the invention.

[0027]FIG. 3 is a schematic view of an embodiment of a flow controllerin accordance with the invention.

[0028]FIG. 4 is a schematic view of another embodiment of a flowcontroller in accordance with the invention.

[0029]FIG. 5 is a schematic view of a third embodiment of a system inaccordance with the invention.

[0030]FIG. 6 is a schematic view of a fourth embodiment of a system inaccordance with the invention.

[0031]FIG. 7 is a schematic view of a fifth embodiment of a system inaccordance with the invention.

[0032]FIG. 8 is a schematic view of a sixth embodiment of a system inaccordance with the invention.

[0033]FIG. 9 is a schematic view of a seventh embodiment of a system inaccordance with the invention.

[0034]FIG. 10 is a plot of peaks for several standard concentrations ofTNT as described in the Examples.

[0035]FIG. 11 is a plot of peak areas as a function of TNT standardconcentration for 118 semi-automated procedure runs on 8 separate daysover a time span of 43 days, as described in the Examples.

[0036]FIG. 12 is a plot of automated calibration data as described inthe Examples.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0037] For the purposes of promoting an understanding of the principlesof the invention, reference will now be made to preferred embodimentsand specific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of theinvention is thereby intended, such alternations and furthermodifications in the invention, and such further applications of theprinciples of the invention as described herein being contemplated aswould normally occur to one skilled in the art to which the inventionpertains.

[0038] The invention encompasses fluid flow systems for separatingmagnetic particles from a fluid medium, and resuspending the particlesone or more times in the same or different media, by controlling therates of fluid flow through a capture zone. In accordance with theinvention, a magnetic field intercepts the fluid flow path in thecapture zone to effect separation. The magnetic field in certainpreferred embodiments is a fixed magnetic field, which term is usedherein to refer to a magnetic field that remains substantially unchangedduring capture and resuspension of magnetic particles. In certainembodiments, the fixed magnetic field is provided by positioning apermanent magnet in a fixed relationship to the fluid flow path. Thisrelationship can be achieved, for example, by affixing the permanentmagnet directly to a conduit defining the flow path, or alternatively byaffixing the permanent magnet to a structure that does not move inrelation to the flow path. In other embodiments, the fixed magneticfield is provided by an electromagnet that is positioned in a fixedrelationship to the fluid flow path and that is configured to generate amagnetic field having a substantially constant field strength duringcapture and resuspension of magnetic particles in an inventive system.

[0039] Inventive systems have extensive utility for performingprocedures which call for not only separation of magnetic particles froma medium, but also the resuspension of the particles in the same ordifferent media in one or more subsequent steps. By controlling theselection of fluids for entry into the fluid flow path and the rates anddirections of fluid flow, inventive systems find advantageous use in awide variety of assays and other techniques that require substrates tobe contacted with various fluids in a predetermined sequence. Thepresent invention is particularly well suited for use in connection withautomated fluid flow systems such as, but not limited to, proceduresreferred to as Sequential Injection Analysis (SIA) procedures. Byautomating an inventive fluid flow separation system, precisemeasurements may be obtained quickly and in a repeatable manner usingrelatively small amounts of reagents and other materials.

[0040] Referring to FIG. 1, system 1 includes fluid flow path 10, havinga first end 20 and a second end 30, and fluid flow controller 50. System1 also includes capture zone 40 between first end 20 and second end 30.Capture zone 40 is distinguished from the remainder of flow path 10 inthat a magnetic field intercepts fluid flow path 10 in capture zone 40.The magnetic field is provided by positioning a magnetic field source 42in a fixed relationship to flow path 10. Controller 50 is effective tovariably impose a positive or negative pressure on the flow path tocause controlled fluid flow through fluid flow path 10 in a first orsecond direction at varying rates. Controller 50 in certain embodimentsis also configured to aspirate media into flow path 10 from differentsources. Flow path 10 can be defined by a tube, as in the embodimentsdescribed in the Examples, or can alternatively be a microchannel.Microchannels, which often have internal diameters of less than about100 microns, can be formed, for example, by machining, microfabricationor molding techniques known in the art. Processes and techniques forforming a microchannel are described, for example, in U.S. Pat. Nos.5,611,214, 5,811,062, 6,129,973, 6,192,596 and 6,200,536.

[0041] In a manner of using system 1, a first mixture is provided in theflow path, the mixture including a plurality of solid magnetic particlesdispersed in a carrier medium. The first mixture, which can also bereferred to as a suspension or a slurry, can be provided in the flowpath in a wide variety of ways, nonlimiting examples of which areprovided in more detail below. Once the first mixture is positionedwithin fluid flow path 10, flow controller 50 exerts a positive ornegative pressure on the mixture to cause the mixture to pass throughcapture zone 40 at a predetermined rate (referred to herein as a“capture rate”) whereby a major portion of the magnetic particles becometrapped in capture zone 40 by the force of the magnetic field, andthereby separated from the carrier medium to form a first magneticparticle isolate. The term “major portion” is used herein to refer to aportion necessary to achieve a desired result in a given separationprocedure within acceptable ranges of precision. For example, wheninventive systems are used to conduct an ELISA assay, examples of whichis described in greater detail below, it is preferred that at leastabout 90% by weight, more preferably at least about 95%, of the magneticparticles are captured by the force of the magnetic field when themixture passes through the capture zone to ensure assay results withinan acceptable range of error. It is to be understood that differentranges of capture efficiency may be preferred when an inventive systemis used in different types of procedures.

[0042] The predetermined capture flow rate is a flow rate sufficientlylow that magnetic attractive forces on the particles in the mixtureexceed viscous and gravitational forces imparted by movement of thecarrier medium. At this flow rate, those particles are held, orcaptured, in the capture zone while the carrier fluid exits the capturezone. For example, in a system described in the example below in whichthe fluid flow path has an internal diameter of about 0.02 inch (˜0.5mm) and the particles are about 0.5 μm in diameter, a preferred captureflow rate is less than or equal to about 13 mm/s (2.5 μl/s). Morepreferably, the capture rate for this example is from about 1.0 to 13mm/s. A person of ordinary skill in the art will readily recognize thatthe preferred rate may vary depending upon a variety of features of agiven system, such as, for example, particle size, particle magneticsusceptibility, magnet field strength, position of the magnetic fieldsource, and flow path configuration (diameter, wall thickness, tubing orchannel material, etc.), and will be able to make adjustments to thecapture flow rate, if necessary, to achieve a suitable degree ofparticle capture.

[0043] Once the magnetic particle isolate is captured, a dispersionmedium is introduced into capture zone 40 and the magnetic particleisolate is perfused with the dispersion medium. After perfusion, thedispersion medium is pulsed through capture zone 40 at a predeterminedrate (referred to herein as a “dispersion rate”) effective to dislodgethe magnetic particle isolate from the capture zone, move the magneticparticles from the magnetic field, and suspend the magnetic particles inthe dispersion medium to provide a second mixture. At the predetermineddispersion flow rate, the higher rate of flow through the fluid flowpath permits viscous forces of the fluid to remove the capturedparticles from the capture zone. An inventive system in certainembodiments is capable of non-destructively separating even fragileparticles, such as, for example, intact blood cells, from a carrierfluid. It is to be understood that the pulse can, in variousembodiments, move the magnetic particles from the capture zone in eitherdirection.

[0044] For example, in a system in which the fluid flow path has adiameter of about 0.02 inches, as described in detail below, a preferredpulse flow rate is from about 250 to about 2500 mm/s (200 μl/s=1018 mm/sin the system described; 500 uL/s=2500 mm/s, 50 uL/s=250 mm/s ). Morepreferably, the pulse rate in a flow path of this diameter is from about750 to about 1250 mm/s. A person of ordinary skill in the art willreadily recognize that the preferred rate may vary depending upon otherfeatures of a given system, and will be able to make adjustments to thedispersion rate to achieve a suitable degree of particle dispersion.

[0045] Where it is desired to also separate the magnetic particles fromthe dispersion medium, the second mixture is then passed through thecapture zone at a predetermined capture rate to again trap a majorportion of the magnetic particles in the capture zone and therebyremoved from the first dispersion medium to form a second magneticparticle isolate. It is readily understood that in a flow path havingonly one capture zone, passing the second mixture through the capturezone will require a reversal of the direction of fluid flow in the fluidflow path. This fluid flow reversal is effected by flow controller 50imposing a reverse pressure on the mixture to cause the fluid to flowthrough the flow path in the opposite direction at a predetermined rate.

[0046] By controlling fluid flow rates through a fixed magnetic field incapture zone 40, as described above, the present invention providessystems that differ from prior art separation/resuspension byeliminating the need for removal of the magnetic field such as byphysical movement of a permanent magnet away from the flow path or,where an electromagnet is used, by turning off the electromagnet oreffecting field fluctuations or reversals. Furthermore, due to preciselycontrolled fluid flow in an inventive system, precise results can beobtained quickly using remarkably small amounts of reagents, samples,selective surfaces and the like. In certain preferred embodiments, thefluid flow path in the capture zone is essentially uninterrupted byscaffolds, rods or other magnetizable matrix structures and theparticles are simply captured against the side of the flow path conduit.In other preferred embodiments, ferromagnetic structures are positionedwithin the flow path to produce high field gradients. Such structuresare often useful, for example, for capturing nanoparticles.

[0047] The composition of carrier fluid and magnetic particles can varydepending on the particulars of the procedure being conducted. Forinstance, many commercial ELISA test kits are available that includemagnetic particles with selective chemistry (immobilized antibodies),along with other reagents for use in a given assay. Thus, in oneembodiment, the carrier fluid and magnetic particles include ELISA assaymaterials. As one representative example, a TNT RaPID Assay™ kit isavailable from Strategic Diagnostics (Newark, Del.) that includesTNT-horseradish peroxidase (TNT-HRP), a color development solution(3,3′5,5′-tetramethylbenzidine and H₂O₂), and a suspension of irregular0.5 μm diameter magnetic particles with immobilized anti-TNT antibody.As another example, particles can be obtained that are covalently linkedto specific oligonucleotide capture probes. Such particles can beutilized for selective purification of nucleic acid fragments from abiological sample. The term “nucleic acid” is used herein to refer toDNA nucleotides and RNA nucleotides, as well as any length polymercomprising DNA nucleotides or RNA nucleotides.

[0048] While the carrier medium described above can be an inert mediumselected solely for transport of magnetic particles to the capture zone,in certain embodiments of the invention, the carrier medium includes atest sample, i.e., a sample in which the presence and/or the quantity ofa specific analyte is to be determined. As such, initial contact of theparticles and the carrier medium begin an incubation period that endsonly when the magnetic particles are separated from the carrier mediumas described above. Of course, in certain assays, such as, for example,a competitive immunoassay, the carrier medium may also include one ormore additional reagents.

[0049] It is readily understood by a person of ordinary skill in the artthat, in order to provide acceptable results, many chemical andbiological assays require one or more wash steps in which unreactedreagents and/or target substances that are not bound to the magneticparticles are washed from the particles. As such, the dispersion mediumin the procedure described above can be a wash solution. It is alsoreadily understood that it is often desirable to conduct multiple washsteps to improve the accuracy of the test or assay. As such, in certainembodiments of the invention, resuspension and recapture of the magneticparticles can be preformed a plurality of times generally as describedabove.

[0050] Many such tests or assays also require suspension of the isolatein a specific reagent (referred to herein as an “analysis reagent”). Forexample, in certain enzyme-bound immunoassay procedures, isolatedsubstances are contacted with or suspended in a substance that producescolor when acted upon by the bound enzyme. As such, certain inventivemethods include perfusing a magnetic particle isolate with a dispersionmedium including an analysis reagent, pulsing the medium through thecapture zone to dislodge the magnetic particle isolate from the capturezone, move the magnetic particles from the magnetic field, and suspendthe magnet particles in the analysis reagent-containing medium. Afterthe suspension is allowed to incubate for a predetermined period oftime, this suspension is then passed through the capture zone to onceagain capture magnetic particles from the suspension, so that one ormore physical and/or chemical properties of the medium containing theanalysis reagent, or the magnetic particle isolate itself, can bemeasured.

[0051] Referring now to FIG. 2, fluid flow system 2 is shown thatincludes a detection zone 60 and detector 62. Detector 62 is in fluidcommunication with, or makes up a portion of, the fluid flow path, andis positioned to detect a physical or chemical property of a fluid inthe detection zone. The detection zone 60 may be separate from thecapture zone 40 (as depicted schematically in FIG. 2), or the detectionand capture zones may partially or completely overlap. The presentinvention contemplates that a wide variety of detectors can be used invarious applications of the invention, including but not limited tooptical detectors, pH detectors, radiation detectors, viscositydetectors and the like.

[0052] The type of detector used in accordance with the invention canvary depending upon the particular type of analysis being performed, andit is well within the purview of a person of ordinary skill in the artto select and configure a suitable detector for use in accordance withthe invention. For example, a variety of assays depend upon thedevelopment of color in an analysis reagent for the identification andquantification of an analyte. For such assays, the detector can be aspectrophotometer or other optical detector configured to pass light orother electromagnetic radiation of a given wavelength through thesolution to measure the degree of color development. As one example, thedetector described in greater detail below includes a U-shapedtransmittance flow cell machined from FEP (McMaster-Carr, Los Angeles,Calif.) and used for absorbance measurements downstream from trappedmagnetic particles. The optical path is 1 cm long and absorbancemeasurements were made using an Ocean Optics (Dunedin, Fla.) fiber opticspectrophotometer and tungsten halogen lamp. A 400 μm quartz fiber wasused for detection. An embodiment including a permanent magnet and anoptical detector as described is set forth in FIG. 8.

[0053] For assays in which added sensitivity is required, one may wishto modify the assay to utilize other detection schemes such aschemiluminescence or liposome-based immunoassays which increasesensitivity by several orders of magnitude. A wide range of labels(i.e., radioactive isotopes, chromogenic or luminescent groups, etc.)can be used and a person of ordinary skill in the art can select andimplement a suitable detector for a wide variety of such assays.

[0054] Fluid flow controller 50 can have a wide variety ofconfigurations in accordance with the invention. In one embodiment,depicted in FIG. 3, flow controller 50 comprises a multi-port selectionvalve 51, a holding coil 54 and a variable speed reversible pump. Inthis embodiment, multi-port selection valve 51 includes a primary port52 and a plurality of secondary ports 53. One of the secondary ports isfluidly connected to the fluid flow path 10, and primary port 52 isfluidly connected to holding coil 54. The other secondary ports 53 areoptionally fluidly connected respectively to sources of various fluidsto be used in a given separation/resuspension procedure. Still otherports can be used for drawing air into the holding coil or fordispelling waste from the system, as discussed in greater detail below.Proximal end 55 of holding coil 54 is fluidly connected to variablespeed reversible pump 58. In one embodiment, pump 58 is a syringe pumpincluding a stepper-motor, which pumps are well known in the pertinentfield. It is of course not intended that the invention be limited tothis type of pump, it being understood that alternative pump designs canbe used as would occur to a person of ordinary skill in the art. Holdingcoil 54 is used as a reservoir for holding a sample, extraction materialor reagents. In each step of a sequential injection procedure, a liquidor slurry is aspirated into the holding coil via a selected valve port,then the valve is switched to the flow path port and the coil contentsare injected into the flow path. Air separators prevent bead slurry,sample, and other solutions from mixing or dispersing in the holdingcoil.

[0055] In another embodiment, depicted in FIGS. 4-6, flow controller 50also includes a multiport valve 57 positioned between and fluidlyconnected to the holding coil and the variable speed reversible pump. Inthis embodiment, proximal end 55 of holding coil 54 is connected to afirst port of the valve, and a second port of the valve is fluidlyconnected to the variable speed reversible pump. A third port of thevalve can conveniently be connected to a source 59 of a wash solution orother reagent for use in flushing and/or cleaning the fluid flow pathupon completion of a given assay. Thus, in this embodiment, after agiven series of separations/resuspensions, valve 57 switches to fluidlyconnect pump 58 and source 59, and pump 58 draws fluid from source 59into reservoir 59 a. After a desired volume of fluid is drawn, valve 57switches again to fluidly connect pump 58 and holding coil 54, and pump58 propels the fluid through the holding coil, selection valve 51 andflow path 10 to thereby flush any remaining magnetic particles and/orreagents therefrom.

[0056] In one manner of performing a separation/resuspension procedureusing this system, one of secondary ports 53 is fluidly connected to asource of magnetic particles in a carrier medium. Selection valve 51 ispositioned to provide fluid communication between primary port 52 andthe particle/medium mixture source, and pump 58 exerts a negativepressure, thereby drawing a quantity of the mixture into holding coil54. Selection valve 51 then moves to provide fluid communication betweenholding coil 54 and fluid flow path 10. After a desired period of timeof further incubation (if additional time is necessary in a givenprocedure), pump 58 exerts a positive pressure, thereby propelling themixture through selection valve 51, into fluid flow cell 10 and throughcapture zone 40. The rate of flow through capture zone is sufficientlyslow to allow a major portion of the magnetic particles in the mixtureto be captured in capture zone 40 by force of the magnetic field,thereby providing a magnetic particle isolate. Where the magneticparticles are derivatized magnetic particles and the carrier mediumincludes a sample to be tested for the presence and/or quantity of aparticular analyte, it is understood that the particles must remain incontact with the medium for a carefully controlled period of time toprovide acceptable assay results. Therefore, in a procedure in whichcontact between the particles and the sample is to be maintained for aspecified length of time, it is important to initiate a timing sequencefrom the time of initial contact thereof.

[0057] In an automated system, the carrier medium is an inert carrier,and the magnetic particles remain separated from the test sample untilmixing occurs in the holding coil or in the fluid flow cell. In thisembodiment, a test sample and other reagents, if needed, are placed,separately or in a premixed solution, in fluid communication with one ormore of the other secondary ports 53.

[0058] Where mixing of the magnetic particles and test sample is tooccur in the holding coil, magnetic particle suspension, test sample andother reagents, separately or in some premixed combination, arealternately drawn into holding coil by coordinated movement of selectionvalve 51 and pump 58. As quantities of each are alternately drawn intoholding coil ( to form a “stacked zone”), the quantities becomeintermixed, thereby initiating an incubation period during “stacking” ofthe holding coil. After the particles/sample/reagents are stacked in theholding coil (without air separation in this case), and optionally aftera desired period of time of further incubation (if additional time isnecessary in a given analysis), selection valve 51 moves to providefluid communication between holding coil 54 and flow path 10, and pumpexerts a reverse pressure on the fluids to move the fluids throughselection valve 51, into flow path 10, and through capture zone at apredetermined capture rate as described above, for separation of themagnetic particles from the other fluids.

[0059] Where mixing is to occur in the flow cell, a magnetic particlesuspension in an inert carrier is first drawn into holding coil bycoordinated movement of selection valve 51 and pump 58, and aspiratedfirst into the flow path and through capture zone at a predeterminedcapture rate to provide a magnetic particle isolate wherein theselective surfaces of the particles are as yet unreacted. Subsequently,test sample and other reagents, separately or in some premixedcombination, are passed into flow path 10 to perfuse the isolate andresuspend the particles. Where a specific order of contact is desiredfor a given analysis, it is of course understood that the order in whichfluids are aspirated into flow path 10 is readily controlled by thecoordinated control of selection valve 51 and pump 58. Furthermore, ifadditional mixing of fluids is desired as an intermediate step of aninventive procedure, it is understood that a resuspended magneticparticle mixture or other fluids can be mixed in holding coil bycreating a “stacked zone” in holding coil 54 by coordinated control ofselection valve 51 and pump 58 generally as described above.

[0060] Once a magnetic particle isolate is formed by separation ofmagnetic particles from the carrier medium, the magnetic particleisolate is perfused with a dispersion medium, such as, for example, awash solution. In certain embodiments, dispersion medium is alreadypresent in reservoir 59 a or present in holding coil 54 “behind” themixture. In other embodiments, such as that depicted in FIG. 4, a sourceof dispersion medium can be accessed by movement of valve 57 to providefluid communication between pump 58 and source 59, aspiration ofdispersion medium into reservoir 59 a, movement of valve 57 to providefluid communication between pump 58 and holding coil 54, and reversal ofpressure applied by pump 58 to cause dispersion medium to flow throughcoil 54, through selection valve 51 and into flow path 10. In suchembodiments, dispersion medium can be aspirated and dispensed into flowcell 10 without further movement of selection valve 51. In otherembodiments, a dispersion medium source is accessible through one ofsecondary ports 53 of selection valve 51. In such an embodiment,dispersion medium is aspirated into flow cell 10 by first drawingdispersion medium into holding coil 54. In such an embodiment fluidcommunication between holding coil 54 and dispersion medium source isprovided by movement of selection valve 51, pump 58 exerts a negativepressure, thereby drawing a quantity of the dispersion medium intoholding coil 54. Selection valve 51 then moves to again provide fluidcommunication between holding coil 54 and fluid flow path 10, and pump58 exerts a positive pressure, thereby propelling the medium throughselection valve 51, into fluid flow cell 10 and through capture zone 40.The rate of flow through the capture zone is initially sufficiently slowto prevent disruption of the magnetic particle isolate. After some ofthe dispersion medium perfuses the isolate, pump 58 exerts a greaterpressure (positive or negative) for a brief period of time to cause afluid flow pulse in the capture zone having a flow rate high enough todislodge the magnetic particle isolate from the capture zone, move themagnetic particles from the magnetic field, and suspend the magneticparticles in the dispersion medium to provide a second mixture.

[0061] The magnetic particles can be separated from the dispersiongenerally as described above and again resuspended and reseparated oneor more times in the same or different media. Alternatively, the secondmixture can be passed to detection zone 60 for detection of one or morephysical or chemical features, or can be collected for furtherprocessing of another type.

[0062] In particularly preferred embodiments of the invention, selectionvalve 51, pump 58 and valve 57 (where present) are under the control ofa pre-programmed sequencer 70 as depicted schematically in FIG. 7.Sequencer 70 controls the movement of valves to provide fluidcommunication between desired flow cells and controls the pump toprovide positive or negative pressure of desired magnitudes atappropriate times, examples of which are discussed above, therebycoordinating the movement of fluids through fluid flow path 10. Infurther embodiments, such as, for example, where sequencer 70 is acomputer, the computer may also be configured to receive informationfrom detector 62 and analyze, tabulate, interpret and/or display suchinformation.

[0063] Sequencer 70 may be comprised of one or more componentsconfigured as a single unit. Alternatively, when of a multi-componentform, sequencer 70 may have one or more components remotely locatedrelative to the others, or otherwise have its components distributedthroughout the system. Sequencer 70 may be programmable, a state logicmachine or other type of dedicated hardware, or a hybrid combination ofprogrammable and dedicated hardware. One or more components of sequencer70 may be of the electronic variety defining digital circuitry, analogcircuitry, or both. As an addition or alternative to electroniccircuitry, sequencer 70 may include one or more mechanical, hydraulic,pneumatic, or optical control elements.

[0064] In one embodiment including electronic circuitry, sequencer 70 isbased on a solid state, integrated microprocessor or microcontrollerwith access to one or more solid-state memory devices (not shown). It ispreferred that this memory contain programming instructions to beexecuted by the microprocessor or microcontroller, and be arranged forreading and writing of data in accordance with one or more routinesexecuted by sequencer 70. In further embodiments, sequencer 70 is anindustrial grade ruggedized programmable personal computer withcustomized software and hardware to practice the present invention. Thispreferred configuration can include communication interfaces such asmodem or network links, and subsystems to accommodate removable media,such as compact disks (CDs) or floppy disks. Although it is preferredthat the processor be readily reprogrammable by software, it may also beprogrammed by firmware, or be configured as an integrated state machine,or employ a combination of these techniques. In yet a further form,sequencer 70 is provided with one or more programmable logic control(PLC) units.

[0065] Any memory associated with sequencer 70 may include one or moretypes of solid-state electronic memory and additionally or alternativelymay include the magnetic or optical variety. For example, the memory mayinclude solid-state electronic Random Access Memory (RAM), SequentiallyAccessible Memory (SAM) (such as the First-In, First-Out (FIFO) varietyor the Last-In First-Out (LIFO) variety), Programmable Read Only Memory(PROM), Electrically Programmable Read Only Memory (EPROM), orElectrically Eraseable Programmable Read Only Memory (EEPROM); anoptical disc memory (such as a CD ROM); a magnetically encoded harddisc, floppy disc, tape, or cartridge media; or a combination of any ofthese types. Also, the memory may be volatile, nonvolitile or a hybridcombination of volatile and nonvolatile varieties.

[0066] Sequencer 70 can also include any control clocks, interfaces,signal conditioners, filters, limiters, Analog-to-Digital (A/D)converters, Digital-to-Analog (D/A) converters, communication ports, orother types of operators as would occur to those skilled in the art toimplement the present invention.

[0067] Use of a flow controller as described above for sequentialinjection offers several advantages for immunoassays. The highlyreproducible timing obtained with sequential injection allows foraccurate analysis that can extend into non-equilibrium measurements, ifdesired, in a very short time frame not generally considered or achievedby a manual technique. Sample volumes injected can be very small, andthere is also very low reagent consumption.

[0068] As stated above, the fixed magnetic field that intercepts capturezone 40 can be provided by a permanent magnet having a fixedrelationship to flow path 10 or by an electromagnet having a fixedrelationship to flow path 10 and generating a substantially constantfield strength. One advantage of permanent magnets is that they do notgenerate heat during operation, which could interfere with the analysis.Where the magnetic field is provided by a permanent magnet, the magnetcan have a wide variety of configurations, including configurationscommonly referred to as a horseshoe configuration or a standard barconfiguration. For example, the permanent magnet or magnets can have arectangular cross-section and in certain embodiments can be glued orfixed by mechanical means to the fluid flow path conduit or to anonmagnetic holding support to form a permanent magnet assembly. Infurther embodiments, the assembly can include a ferromagnetic harness tohouse the magnet or magnets and to intensify and focus the magneticfield. The magnets are preferably oriented with their magnetic lines offorce perpendicular to the longitudinal axis of the flow path. Alternatecross-sectional shapes, orientations, and magnetic pole orientation withrespect to the container are also envisioned.

[0069] In addition, permanent magnets can have a variety ofcompositions. Permanent magnets of rare earth alloys having a surfacefield strength in the range of several hundred Gauss to severalkilo-Gauss are preferred. In one embodiment, the permanent magnet is ahigh energy permanent magnet made from Neodymium-Iron-Boron orSamarium-Cobalt. Such magnets, and a wide variety of alternativepermanent magnets suitable for use in connection with the invention areavailable commercially. A wide variety of such configurations andcompositions are included within the meaning of the term “permanentmagnet” as used herein.

[0070] In a standard bar magnet, gradients exist because the magneticfield lines follow non-linear paths and “fan out” or bulge as they movefrom North to South, as depicted schematically in FIGS. 6 and 8. Theseeffects typically create gradients of about 0.1 to 1.5 kGauss/cm in highquality laboratory magnets. While it is not necessary to the invention,a person of ordinary skill in the art will recognize that thesegradients can be increased by manipulating the magnetic circuits so asto compress or expand field line density. For example, if the gradientat one pole of a bar magnet is of insufficient strength, moving a secondbar magnet with an identical field in opposition to the first magnetwould cause repulsion between the two magnets. The number of field lineswould remain the same, but they would become compressed as the twomagnets were forced closer together. Thus, an increased gradient wouldresult. The addition of magnets of opposing field to this dipoleconfiguration to form a quadrupole could further increase the size ofthe region of high gradients. Other configurations such as adjacentmagnets of opposing fields would also create gradients higher than thoseseen in a bar magnet of equivalent strength. Yet another method ofincreasing gradients in external field devices is by adapting the polepiece design. For example, if the configuration of a standard dipolemagnet were changed by making one of the magnets into a pointed magnet,all field lines would flow towards the point, dramatically increasingthe gradient around that region.

[0071] In accordance with the invention, the magnetic field in thecapture zone of the fluid flow path has a strength that will capturemagnetic particles selected for use in accordance with the invention ata relatively low flow rate (i.e., a flow rate of from about 1 to about13 mm/s), and that is not great enough to retain the particles in afluid flow having a relatively high flow rate (i.e., a flow rate of fromabout 250 to about 2500 mm/s). It is readily understood that thepreferred field strength may vary depending upon a variety of factors,including, but not limited to, the inner dimensions of the flow cell inthe capture zone and at other locations, the size and susceptibility ofthe magnetic particles used in a given procedure, and the desired ratesof separation and perfusion if a given procedure calls for such. It iswell within the purview of a person of ordinary skill in the art, inview of the present specification, to select a field strength and toselect a suitable magnetic field source, whether of the permanent magnetor electromagnet variety, for a wide variety of inventive systems. Inone preferred embodiment, the capture zone includes average magneticfield gradients of 0.1 to 2 kGauss/cm. In another preferred embodiment,ferromagnetic materials are placed within or in close proximity to theflow path to produce localized regions of high magnetic field gradientfor capturing smaller, less magnetic particles.

[0072] Turning now to the magnetic particles themselves, the term“magnetic particle” is used herein to refer to a particle that isresponsive to a magnetic field. Magnetic particles selected for use inaccordance with the invention preferably include a ferromagneticmaterial, more preferably a superparamagnetic material.Superparamagnetic materials, regardless of their size (i.e., oftenranging from 25 nm to 100 microns,) have the property that they are onlymagnetic when placed in a magnetic field. Once the field is removed,they cease to be magnetic and can normally be dispersed into suspension.The basis for superparamagnetic behavior is that such materials containmagnetic material in size units below 20-25 nm, which is estimated to bebelow the size of a magnetic domain. A magnetic domain is the smallestvolume for a permanent magnetic dipole to exist. Hence, these materialsare formed from one or more or an assembly of units incapable of holdinga permanent magnetic dipole. Such magnetic particles are commonly ofpolymeric material containing a small amount of ferro-magnetic substancesuch as iron-based oxides, e.g., magnetite, transition metals, or rareearth elements, which causes them to be captured by a magnetic field.The magnetic material of choice is magnetite, although other transitionelement oxides and mixtures thereof can be used.

[0073] For many preferred applications of the invention, the surface ofmagnetic particles is coated with a ligand or receptor, such asantibodies, lectins, oligonucleotides, or other bioreactive molecules,which can selectively bind a target substance in a mixture with othersubstances. The surface chemistry of the magnetic particle can also beused to capture target substances due to electrostatic interactions, vander Waals interactions, dipole-dipole interactions or hydrogen bondinginteractions between the target substances and the surfaces of themagnetic particles. Magnetic particles have been used for variousapplications, particularly in health care, e.g. immunoassay, cellseparation and molecular biology, and many such particles are availablecommercially. Superparamagnetic particles useful for performing suchprocedures should provide for an adequate binding surface capacity forthe adsorption or covalent coupling of one member of a specific affinitybinding pair, i.e., ligand or receptor.

[0074] Some magnetic particles are composed of spherical polymericmaterials into which has been deposited magnetic crystals. Thesematerials, because of their magnetite content and size, are readilyseparated in relatively low fields (0.5 to 2 kGauss/cm) which can easilybe generated with open field gradients. Another similar class ofmaterials are particles that typically are produced in the size range offrom about 0.5 to about 0.75 microns. Another class of suitable materialincludes particles that are basically clusters of magnetite crystals,about 1 micron in size, which are coated with amino polymer silane towhich bioreceptors can be coupled. These highly magnetic materials areeasily separated in relatively low gradients (i.e., gradients as low as0.5 kGauss/cm) and, due to their size, they can remain suspended forextended periods of time.

[0075] The preferred diameter of a magnetic particle used in accordancewith the invention is in the range between 0.1 to 300 microns. Magneticparticles are today widely available commercially, with or withoutfunctional groups capable of binding antibodies or DNA molecules orcontaining other binding sites for sample purification. Suitableparamagnetic particles are commercially available from Dynal Inc. ofLake Success, N.Y.; PerSeptive Diagnostics, Inc., of Cambridge, Mass.;and Cortex Biochem Inc., of San Leandro, Calif.

[0076] Another type of magnetic material that can be used in accordancewith the invention are nanosized colloids (see, for example, U.S. Pat.No. 4,452,773 to Molday, U.S. Pat. No. 4,795,698 to Owen et al, U.S.Pat. No. 4,965,007 to Yudelson; and U.S. patent application Ser. No.07/397,106 by Liberti, et al). Nanosized colloids are typically composedof single to multicrystal agglomerates of magnetite coated withpolymeric material which render them aqueous compatible. Individualcrystals can range in size from about 8 to about 15 nm. The coatings ofthese materials have sufficient interaction with aqueous solvent to keepthem in the colloidal state almost, if not, permanently. A person ofordinary skill in the art will recognize that, because of their size andinteraction with solvent water, substantial magnetic gradients arerequired to separate nanosized colloids. Therefore, in a system in whichsuch colloids are used, magnetic fields of greater strength arepreferred.

[0077] One advantageous feature of the invention is that the time duringwhich magnetic particles are suspended in a given reagent or othermedium can be precisely controlled. In addition, the inventive approachpermits the implementation of an automated immunoassay that is quick andsensitive in the micromolar concentration range. A particularlyadvantageous use of the present invention is for the performance of anELISA assay using an automated sequential injection procedure, whichutilizes surface-derivatized magnetic particles to determine the amountof analyte in a test sample by competitive immunoassay.

[0078] Competitive immunoassays with magnetic-particle-bound antibodiesand enzyme-amplified detection involve several steps. Using TNT as theanalyte, for example, the first step is to mix the TNT sample, theTNT-enzyme conjugate, and immobilized antibodies. In a semi-automatedprocedure in accordance with the invention, these ingredients can bemixed, for example, in a polypropylene vial for competitive binding. Theuse of antibodies that are immobilized on magnetic particles facilitatesseparation of the solution components from the antibody-bound componentsin subsequent steps. Once the particles have been separated and washed,an analytical color development solution is added. This containsreagents for enzyme-catalyzed conversion of a substrate to a coloredproduct for detection. After a precise reaction time, an absorbencymeasurement is made.

[0079] A typical sequence is as follows: (1) magnetic particles that arecoated with antibodies against the analyte are mixed with the testsample including the analyte and an analyte-enzyme conjugate in whichthe enzyme acts upon a coloring reagent to produce color, and incubatedfor a predetermined period of time; (2) this mixture is aspirated intothe fluid flow cell and through a capture zone in a fixed magneticfield, whereupon the magnetic field traps the particles in the flow pathto form a magnetic particle isolate; (3) the magnetic particle isolateis resuspended in wash solution one or more times by aspiration andpulsing of a wash solution, with alternating recapture in the capturezone by a flow reversal and passage of the resuspended particles throughthe capture zone at a capture flow rate; (4) the washed magneticparticle isolate is resuspended in a coloring reagent by aspiration andpulsing of the reagent; (5) after a reaction period of a predeterminedlength, the magnetic particles are recaptured in the capture zone by aflow reversal and passage of the reagent and particles through thecapture zone at a capture flow rate; (6) the coloring reagent is passedthrough an optical cell for detection of color development; and (7) awash solution is passed through the flow cell at a relatively high rateto wash any remaining particles and/or reagents from the flow cell tocondition the system for a subsequent procedure.

[0080] As such, in one embodiment, the present invention provides anautomated ELISA instrument that requires minimal operator intervention.The automated ELISA system works like the manual procedure, but withsmaller reagent volumes and shorter reaction times, and thus can beconfigured to provide a field-portable instrument capable of reliablyand reproducibly analyzing test sample. The method is versatile andflexible and may therefore also be adapted to many differentapplications. The spent magnetic particles may be discharged after eachanalysis. This eliminates the problems of instability of reactionsurfaces and eliminates the need for additional time traditionallyrequired for regeneration of the solid-reacting phase in order to notonly save time and increase sampling frequency but also to provide eachindividual sampling cycle with a fresh, uniform portion of magneticparticles. The spent magnetic particles are collected off line and maybe regenerated later. The small volumes required, reduced samplehandling, and precise reproducibility of inventive procedures have beenshown to be useful for improving cumbersome, time-consuming, laborintensive, and semiquantitative traditional immunoassays.

[0081] In addition to the above-described embodiments, a variety ofalternative configurations of the described systems are within the scopeof the invention. As one example, the present invention contemplates asystem in which the flow path includes multiple capture zones. In suchan embodiment, a procedure could be performed in which fluid flow in thefluid flow path is unidirectional. In a system with multiple capturezones, magnetic particles can be separated from a carrier mediumgenerally as described above in a first capture zone to provide a firstmagnetic particle isolate. The first isolate could then be resuspendedby a pulse of a dispersion medium in the direction of a second capturezone to provide a second mixture. Flow could then be stopped if desiredto provide a period of incubation between the capture zones, and thenresumed in the same direction to pass the second mixture through thesecond capture zone to separate the magnetic particles from thedispersion medium and thereby form a second magnetic particle isolate.Further resuspension/recapture steps could then be performed, in furthercapture zones on the fluid flow path, or in the same capture zones byalso utilizing fluid flow reversals. Such a system can be used, forexample, when several capture/resuspension steps are necessary for agiven assay or other procedure.

[0082] In another embodiment, multiple assays can be performed on thesame test sample by including reagents for multiple assays in fluidconnection with secondary ports 53 of multiport selection valve 51 andsequentially running different assays on quantities of the same sample.For example, it may be desired to perform one assay on a sample, andthen perform a different analysis on the same sample. In thisembodiment, after completion of an assay and flushing the system with awash solution or other cleansing reagent, the system can be configuredto automatically mix the test sample with magnetic particles havingdifferent selective groups bound thereto, and perhaps other reagents tosequentially perform another assay.

[0083] Additional modifications are envisioned that address specificrequirements of certain assays. For example, it is understood thatcertain color-development solutions undergo photocatalysis when exposedto light. As such, in embodiments that utilize a color developmentreagent, conduits defining the fluid flow path and other fluid transportpathways through which a coloring reagent passes are made of an opaquematerial or covered with an opaque material, such as, for example, alayer of tape, to prevent exposure of the coloring reagent to light orother electromagnetic radiation. It is also understood that tape orother opaque material can also be used to cover flow lines that mightcontain or transport other light-sensitive reagents.

[0084] Similarly, it is understood that certain uses of the inventioninvolve materials, including but not limited to DNA, protein andcombinations thereof, that are temperature sensitive. As such, aninventive system can optionally include temperature control for samplehandling of biomolecules. Temperature control is useful for optimizingbinding and elution rates for DNA hybridization and elution, as well asfor DNA amplification using polymerase chain reaction (PCR) or otherenzyme amplification methods requiring thermal cycling. Elevatedtemperature can help to purify a sample by excluding interferents eitherduring analyte extraction from a sample or during a subsequent washstep. It is well within the purview of a person of ordinary skill in theart to include temperature control elements in an inventive apparatusand to perform inventive procedures at predetermined, controlledtemperatures.

[0085] The invention will be further described with reference to thefollowing specific Examples. It will be understood that these Examplesare also illustrative and not restrictive in nature.

EXAMPLES Example One Sequential Injection Analysis of TNT in Water usingan Enzyme-Linked Immunosorbent Assay

[0086] An experiment was conducted to demonstrate capture andresuspension of magnetic particles in an inventive system as follows:

[0087] Chemicals: TNT RaPID Assay™ kits provided the ELISA reagents(Strategic Diagnostics, Newark, Del.). The reagents used were aTNT-horseradish peroxidase (TNT-HRP) conjugate, a color developmentsolution (3,3′5,540 -tetramethylbenzidine and H₂O₂), and the suspensionof irregular 0.5 μm diameter magnetic particles with immobilizedanti-TNT antibody. The color development reagent solution contained thechromogenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB) and H₂O₂.Exact formulations are proprietary, but optimized reagent formulationshave been published for similar atrazine ELISAs using HRP-atrazineconjugate and TMB. C. S. Hottenstein, F. M. Rubio, D. P. Herzog, J. R.Fleeker and T. S. Lawruk, “Determination of trace atrazine levels inwater by a sensitive magnetic particle-based enzyme immunoassay”, J.Agric. Food Chem., 44(11), (1996) 3576-3581.

[0088] Wash solution consisted of pH 6.2 0.033 M phosphate, 0.033 MNaCl, and 0.1% Polysorbate 20 (P20) (BiaCore, Piscataway, N.J.)non-ionic surfactant. Wash solution also served as carrier solution inthe flow system. DNA Zap™ solutions were from Ambion (Austin, Tex.). Astandard solution of TNT from Supelco of 1000 micrograms/ml TNT inacetonitrile was diluted to prepare TNT standards in pH 6.2 0.033 Mphosphate, 0.033 M NaCl and 0.007% P20.

[0089] Apparatus: The flow system set forth in FIG. 9 was used, in whicha FiaLab 3000 system (FIAlab Instruments, Inc., Bellevue, Wash.)provided the stepper-motor-driven syringe pump with a built-in 3-wayvalve (Cavro, Sunnyvalve, Calif.) and a 10-port Cheminert® selectionvalve (Valco, Houston, Tex.). The wash inlet, color inlet, and wasteoutlet were 0.030 inch i.d. fluorinated ethylene-propylene (FEP) tubing(Valco). The color inlet was shielded from light by shrink wrap tubing.The holding coil and cell outlet were also shielded from light. The 600μl holding coil was 0.030 inch i.d. polyetheretherketone (PEEK) fromUpchurch Scientific (Oak Harbor, Wash.). The flow line contacting themagnet, extending from the valve to the optical cell was 0.02 inch i.d.PEEK. The volume from the valve to the center of the magnetic zone was22 μl, and from the center of the magnet to the optical cell was 18 μl.

[0090] A ½×¼×⅝ inch NbFeB permanent magnet was obtained from DexterMagnetic Technologies (Windsor Locks, Conn.). The ¼×⅝ inch faces of themagnet were its poles. One pole was placed against the magnetic zonetubing, with the tubing running along the ⅝ inch length. The tubing hadbeen thinned to create a flat surface for the magnet by filing one sideof the 0.062 inch outside diameter 0.02 inch inside diameter PEEK tubeuntil the total thickness was 0.045 inch. In principle, this leaves0.004 inch of tubing wall between the channel and the magnet; howeverthe actual distance may have been somewhat larger or smaller due tovariations in the centering of the channel in the tubing.

[0091] A transmittance cell detector was drilled out of FEP(McMaster-Carr, Los Angeles, Calif.). As shown in FIG. 9, the inlet,outlet, and optical path of the detector formed a U-shaped channel. Theinlet and outlet were tapped to accommodate Upchurch tube fittings. Theflow channel through the optical path was 1 cm long and 1 mm indiameter. The illumination fiber was 400 μm and the collection fiber was200 μm. Both fibers were quartz contained in 0.062 inch outside diameterstainless steel at the ends. Fiber ends were friction-fitted into theflow cell. The transparency of the FEP allowed the flow channel to bevisible during method development. During actual runs, the cell wascovered with tape to prevent photocatalysis of the color-developmentsolution. The LS-1 tungsten halogen lamp and SD-2000 CCD arrayspectrometer were obtained from Ocean Optics (Dunedin, Fla.). A type NG5optical filter (Schott Glass Technologies, Inc., Duryea, Pa.) wasnecessary to attenuate the lamp. With this filter, the integration timeof the CCD was set at 75 milliseconds per scan to maintain a safe marginaway from light saturation at 630 nm. Absorbency was monitored atλ_(max) of the enzyme product, 630 nm, relative to the baselinemonitored at 730 nm to correct for instrument variations and refractiveindex mixing in the optical path.

[0092] System software was written using VisualC++ (Microsoft, Redmond,Wash.) and the OOIWinIP dynamic link library (Ocean Optics) forcontrolling the CCD spectrometer.

Methodology, Semi-automated Procedure

[0093] Fluid and Magnetic Particle Handling. The competitive immunoassayon magnetic particles, with enzyme-catalyzed amplification, entailedseveral fluid and particle handling steps. These are described insequence below for a semi-automated procedure in which the competitiveassay is set up in a vial by addition of 100 μL of analyte-containingsample, 200 μL of a suspension of magnetic particles withsurface-immobilized antibodies, and 100 μL of analyte-enzyme conjugatein that order.

[0094] The semi-automated approach to be described begins after thecompetitive assay mixture has been mixed in the test vial. Theinstrument times the incubation period so that manual timing is notnecessary, and the tedious manual separations are replaced by automaticparticle capture in a magnetic flow cell, with fluidic delivery of washsolutions and the subsequent color development solution. Colordevelopment reaction time is no longer dependent on manual timing and nostop solution need be used. Instead, the instrument automaticallydelivers the colored product to the detector after a precise time.

[0095] In the semiautomated procedure, initial manual steps were used toinject TNT solution, magnetic particle suspension, and enzyme conjugate,in that order, into a polypropylene vial followed by 10 seconds ofmixing. After an incubation time of 4 minutes, the sequential injectionmethods described in Table 1 were executed, using the sequentialinjection system shown in FIG. 9. For purposes of clarity, standardimplementation details were omitted, namely the use of 10•L airseparators between fluids, and loading of fluids into the holding coilprior to each injection. At least 0.5 seconds of delay are scheduledafter each syringe move to allow for pressure equilibration beforeswitching the selection valve. This helps to maintain the air segmentsat atmospheric pressure. The carrier solution in the flow system wasidentical to wash solution. A small air segment is preferably used toisolate reagents from carrier solution in the flow controller, however,care is taken not to perfuse the final portion of a reagent and airseparator through the flow path because air bubbles interfere withoptical detection.

[0096] Programmed steps were used to pull the competitive assay mixtureinto the system, capture the magnetic particles having a mixture of TNTand enzyme conjugate bound to the anti-TNT antibodies, wash theparticles, mix the particles with reagents for enzyme-catalyzedgeneration of a colored product, hold the particles for a precise colordevelopment time, recapture the particles, and deliver the coloredsolution through a transmittance cell for absorbency measurement. Thepeak area of the absorbency measurement was used for quantification.Table 1 describes the standard reference semi-automated procedure ingreater detail. The color development time in this procedure was 4minutes. The total time for one run was 17.2 minutes, from the beginningof the mixture incubation time to the completion of the absorbencymeasurement, discarding the particles, and clearing the mix and colorinlets with air. This compares to an assay time of over 40 minutes forthe manual procedure. TABLE 1 Sequential Injection ELISA Procedure. FlowVolume, Rate, Procedural Step Solution Port μl μl/s 1 Initialize mixbeads, enzyme, mix inlet sample 2 Aspirate mix into beads, enzyme, mix325 40 the holding coil sample 3 Inject mix to the cell beads, enzyme,cell −320 2.5 and capture beads sample 4 Disperse beads in 5 beadssuspended cell 5 200 μl in enzyme + sample 5 Discard residual mix Wash,air mix −100 200 and air segment in the separator holding coil into themixture inlet 6 Disperse beads in Beads suspended cell 15 200 15 μl inenzyme + sample 7 Capture and wash Wash cell −115 2.5 the beads wash 8Disperse beads in Beads suspended cell 15 200 15 μl of wash solution inwash 9 Capture and wash Wash solution cell −115 2.5 the beads 10Disperse beads in 5 Beads suspended cell 5 200 μl in wash 11 Initializecolor inlet 12 Aspirate into the Color reagent color 200 40 holding coil13 Inject color reagent Color reagent cell −80 2.5 while capturing beads14 Disperse beads in Beads suspended cell 15 200 15 μl in color reagent15 Incubate for colorTime minutes 16 Capture beads while Reacted colorcell −157 1 inject color peak reagent to the optical cell 17 Discard towaste Wash, air, color, cell −240 200 through flow cell beads 18 Restorethe color inlet 19 Restore the mix inlet 20 Rinse the holding Wash cell−80 50 coil

[0097] Initial conditions and inlet line preparation. The inlets at thebeginning of an assay run were set up to contain 20 μL of wash solutionextending out from the valve port (which helps to prevent unwanted pickup of air during valve switching) with the remainder of the tubecontaining air. To initialize a port before pulling solution into theholding coil, the volume of the tube (previously measured and aparameter in the program) plus 5 μL was pulled into the holding coil andthen this volume plus an additional 30 μL of wash solution was expelledto waste. This fills the inlet tube with solution. After an assay, themix and color ports were restored to the initial conditions.

[0098] Competitive mixture handling and magnetic particle capture.Before pulling in mix or color solution, a 10 microliter air segment waspulled into the holding coil. For the competitive mixture solution, 325μL of the mixture was pulled into the holding coil at 40 μL/s using the10 μl air segment to separate the mixture from the carrier solution. 320μL of the sample were then pushed forward at 2.5 μL/s to capturemagnetic particles on the tubing wall of the magnetic capture cell. Theair segment and trailing wash solution were discarded out the mix portat 200 μL/s. Velocities less than 200 μL/s were not reliable forremoving air from FEP tubing walls in 0.03 inch i.e. tubing. Thepresence of at least 0.007% of a surfactant such as P20 prevents breakupof air on hydrophobic surfaces.

[0099] Washing steps. Captured magnetic particles were washed by firstresuspending them in solution using a 15 μL pulse at 200 μL/s thatpulled the particles back toward the multiport valve. Thus, the particlesuspension resided in the 22 μL zone between the valve and the magneticcapture zone. The particle suspension was then pushed back to themagnetic capture zone at 2.5 μL/s, capturing the particles and perfusingthem with wash solution. The wash step pushed 115 μL of solution in theforward direction. There were two such wash steps in the standardprocedure. The first of these steps completes pushing the competitivemixture solution through the magnetic cell followed by wash solution,while the second such step resuspends the particles in wash solution,recaptures, and perfuses them with wash solution.

[0100] Color development and detection. The particles were resuspendedbetween the magnetic cell and the valve with a 5 μL pulse at 200 μL/s.The color port was initialized and an air segment was set up asdescribed above; then 200 μL of color development reagent solution waspulled into the holding coil. 80 μL of color development reagent waspushed from the holding coil toward the flow cell at 2.5 μL/s torecapture the magnetic particles and perfuse them with solution. Thenthe particles were resuspended in reagent solution using a 15 μL pulseat 200 μL/s, again leaving the particles in the 22 μL zone between thevalve and the magnetic capture zone. The particles resided here for thecolor development time, after which they were automatically recapturedin the magnetic cell while the solution was pushed through thetransmittance optical cell at 1 μL/s. (The latter step used a 157 μLinjection, using most of the color development reagent solutionremaining to push the color peak through the optical cell.)

[0101] Wash and restoration steps. At the end of each semiautomated run,the inlet lines for the mix and color ports were cleared by pushing airout. Between runs, the inlets were set up to starting conditions asdescribed above, and the holding coil was rinsed by expelling additioncarrier (wash) solution.

[0102]FIG. 10 shows peaks for several standard concentrations of TNT. Inthis competitive assay, no sample TNT leads to the largest peak andincreasing TNT concentrations lead to smaller peaks. The 527 and 5000ng/ml runs were not distinguishable and were taken as ∞ concentration.Note that the ∞ peak area was non-zero. Runs performed using buffersolution in place of TNT-HRP conjugate resulted in peak areas equal tothe ∞ peak area of the semi-automated method, proving that the ∞baseline was not due to residual enzyme activity. Particles contain ironspecies which should be responsible for some dye formation via theFenton reaction in the color development solution that contained H₂O₂.There was no peak when buffer solution replaced particle suspension in ablank run.

[0103]FIG. 11 shows peak areas as a function of TNT standardconcentration for 118 semi-automated procedure runs on 8 separate daysover a time span of 43 days. These illustrate the expected sigmoidalshape of the calibration curve and the stability of the calibration overa long time period.

[0104] The absorbency peak areas, B, for this type of immunoassay dataare customarily fitted to one of the sigmoidal relationships inequations 1 and 2. Fare, T. L.; Sandberg, R. G.; Herzog, D. P.;“Considerations in immunoassay calibration”; In EnvironmentalImmunochemical Methods; Emon, J. M. V., Gerlach, C. L., Johnson, J. C.;American Chemical Society: Washington, D.C., 1996; ACS Symposium Series646; pp. 240-253.

B/B _(o)={1+(C/C _(0.5))^(b)}⁻¹  (1)

(B−d)/(a−d)={1+(C/C _(0.5))^(b)}⁻¹  (2)

[0105] The parameter B_(o) gives the peak area of the blank, so B/B_(o)is the sample to blank peak area ratio used as a measure of response. Cis the sample concentration and C₀₅ is the concentration where B/B_(o)is 50%. The parameter “b” is a fitting parameter related to the slope ofthe linear region of the curve. When the peak area at infiniteconcentration is nonzero, as is the case in FIG. 10, the relationship inequation 2 is used. The left side is a “corrected” B/B_(o). Theparameter “d” is the fitted value for the peak area of the infiniteconcentration peak and “a” is the fitted value for B_(o). FIG. 11 plotsthe fitted curve for these data obtained using the semi-automatedprocedure.

[0106] Our results are in very reasonable agreement with the vendors,especially considering the recognized uncertainties in fitting sigmoidalcalibration curves in immunoassays. Our procedure mixes antibody (onmagnetic particles) and TNT-HRP conjugate in the same ratio as thevendors recommended procedure. Our fit gives a value of 1.51 ng/ml forthe TNT concentration where the peak area is 50% of the blank peak area(corrected for the infinite concentration peak area), compared to avalue of 1.44 in the vendor's literature. The TNT concentration wherethe peak area is 90% is 0.16 ng/ml, compared to 0.07 claimed by thevendor. The vendor uses this criterion to define a lower detectionlimit, as have others. C. S. Hottenstein, F. M. Rubio, D. P. Herzog, J.R. Fleeker and T. S. Lawruk, “Determination of trace atrazine levels inwater by a sensitive magnetic particle-based enzyme immunoassay”, J.Agric. Food Chem., 44(11), (1996) 3576-3581.

[0107] The standard deviations and relative standard deviations for eachtest concentration are given in Table 3. TABLE 3 Standard deviations andrelative standard deviations at test concentrations for thesemi-automated approach. Average Standard Relative Number Concentration,Peak Area, Deviation, Standard of Ng/ml Absorbance Absorbance Deviation,% Points Blank 0.1087 0.0099 9.1 38 0.4681 0.0849 0.0057 6.7 14 0.9170.0736 0.0049 6.7 12 1.817 0.0567 0.014 25 7 4.265 0.0350 0.0027 7.7 109.345 0.0270 0.0025 9.4 8 18.76 0.0212 0.00073 3.4 8 47.55 0.0143 0.001711.8 4 110.4 0.0138 0.0012 8.9 4 527.1 0.0120 0.00096 8.0 5 5000 0.01130.00048 4.3 5

[0108] Two standard deviations from the mean of replicate blank runs isanother measure used for defining detection limits, C. S. Hottenstein,F. M. Rubio, D. P. Herzog, J. R. Fleeker and T. S. Lawruk,“Determination of trace atrazine levels in water by a sensitive magneticparticle-based enzyme immunoassay”, J. Agric. Food Chem., 44(11), (1996)3576-3581, and is reported to be an industry standard method. Deshpande,S. S. Enzyme Immunoassays: From Concept to Product Development; Chapman& Hall: New York, 1996, p. 323. The vast majority of our data for blankand standard runs is within two standard deviations of the mean. Usingthis criterion, our data yield a value of 0.4 ng/ml for the detectionlimit. It is again worth noting our results are based on data taken over43 days, not replicate runs in a single day.

[0109] A 43 day reproducibility test is quite severe. For this TNT assaykit, the vendor reports %CV values within assays and between assays of3-8% for 5 replicates on each of 5 days (25 samples), and specifies a%CV of 10%. %CV is the coefficient of variation (standard deviation/meantimes 100), a term for relative standard deviation used in theimmunoassay literature. Deshpande, S. S. Enzyme Immunoassays: FromConcept to Product Development; Chapman & Hall: New York, 1996, p. 314.Our results (Table 3) with values typically 6 to 9% over 43 days arequite good and in reasonable agreement with vendor specifications. Oneconcentration (1.8 ng/ml with a standard deviation of 25%) is clearly anoutlier, while a couple of concentrations are at 3 to 4%. For additionalcomparison, in an ELISA assay for alachlor using an automated microplatesystem, Young et al. (Young, B. S.; Parsons, A.; Vampola, C.; Wang, H.;“Evaluation of an automated immunoassay system for quantitative analysisof atrazine and alachlor in water samples”; In EnvironmentalImmunochemical Methods; Emon, J. M. V., Gerlach, C. L., Johnson, J. C.;American Chemical Society: Washington, D.C., 1996; ACS Symposium Series646; pp 183-190) report %CV values ranging from 2 to 9% for duplicatestandards on each of five days. For triplicate spiked Milli-Q watersamples on five separate days, their %CV values were over 7%. %CV valuesof 5 to 10% for replicates over 5 days have been reported for a magneticparticle based immunoassay for atrazine. C. S. Hottenstein, F. M. Rubio,D. P. Herzog, J. R. Fleeker and T. S. Lawruk, “Determination of traceatrazine levels in water by a sensitive magnetic particle-based enzymeimmunoassay”, Journal of Agricultural and Food Chemistry, 44(1996)3576-3581. We conclude that our precision over 43 days was very good.

Comparison TO Manual Procedures

[0110] The procedure above was designed to work like the manualprocedure, but with smaller reagent volumes and shorter reaction times.The ratio of antibodies to enzyme conjugate was the same as in themanual procedure, but we used 200 μL of particle suspension and 100 μLof TNT-HRP conjugate in contrast to volumes of 500 and 250 μL in themanual method. The incubation time and color development times of 4minutes each are significantly shorter than the manual method procedureusing a 15 minute incubation time and a 20 minute color developmenttime. Our shorter times were sufficient to obtain adequate peak areas,while shorter times would not have significantly shortened overallanalysis time.

[0111] Automation reduced variability due to inconsistencies that occurin manual procedures, such as, for example, incubation time and colordevelopment time. The present work shows that the semi-automated andautomated procedures exhibit excellent results over a time period of 43days.

Analysis OF Color Development Data

[0112] Increasing color development time increases product generation asexpected, as shown in the data for blank runs (under the standardreference semi-automated procedure) as a function of color developmenttime in Table 4. TABLE 4 Effect of Color Development Time Time, blankpeak min area, 0.5 0.0157 1 0.0335 2 0.0647 4 0.109 8 0.196

[0113] One could obtain greater peak areas with longer developmenttimes, but there is no apparent advantage to doing so. It should benoted that in the manual procedure, 500 μL of color development solutionare used, and this is diluted with an additional 500 microliter of stopsolution. The colored product is evenly distributed in 1 ml. In the flowsystem, the magnetic particles with conjugated enzyme are located withina 22 microliter tubing zone and the colored peak passes through theoptical cell in approximately 50 μL of pumped volume. Undoubtedly somedispersion occurs on going from the 0.02 inch tubing to the 1 mm (ca.0.04 in) flow cell diameter. Thus, the colored product is at least 20time more concentrated than in the manual procedure. In addition, wehave observed that the blue colored product at neutral pH has a higherabsorbency peak than the yellow form in acidic solution.

[0114] Increasing incubation time increases peak areas for both blankand standard samples, as shown in Table 5. TABLE 5 Effect of CompetitiveMix Incubation Time Time, Blank Peak Std Peak min Area, B₀ Area, B B/B₀0.5 0.0765 0.0289 0.378 1 0.0782 0.0308 0.394 2 0.0948 0.0351 0.370 40.109 0.0350 0.322 8 0.160 0.0470 0.294 16 0.224 0.0531 0.237

[0115] The response in terms of B/B_(o) decreases somewhat with time atincubation times longer than 1 minute, as shown in Table 5. For example,for the 4.3 ng/ml sample used, the B/B_(o) value changed from 0.32 to0.29 from 4 to 8 minutes in these runs. The slope does not change from 4to 16 minutes. Since the assay does not reach equilibrium within thevendor recommended 15 minute incubation time, the selection ofcompetitive mixture time is somewhat arbitrary. Increasing use ofnonequilibrium assays has been noted. Deshpande, S. S. EnzymeImmunoassays: From Concept to Product Development; Chapman & Hall: NewYork, 1996, p. 309. Failure to control the incubation time can influencethe reproducibility of the analyses. In our semi-automated assay, theincubation period is automatically timed.

[0116] We examined a number of other factors that could influence thereproducibility of the calibrations. Shielding the color developmentsolutions from light throughout the procedure made a measurabledifference in analytical peak areas and was important forreproducibility. Room lighting was demonstrated to have an effect onassay baselines by photoreaction of the color development solution whenoptically clear tubing was used. The antibody-derivatized magneticparticles, TNT-enzyme conjugate, and color development reagent solutionswere kept at 0° C. as a precaution, although this was not necessary. Thereagent vendor actually states that the enzyme conjugate and particlesmust be allowed to reach room temperature before use. As a test, enzymeand antibody solutions were deliberately left at room temperature for8.25 hours and used at room temperature. Peak areas for blank and 4.265ng/ml TNT were well within the margin of error for the calibrationrepeatability runs to be described below and plotted in FIG. 11. (Thisfigure actually contains these room temperature runs.)

Example Two Cleaning of Apparatus Between Assays

[0117] Carryover cleaning. To eliminate carryover from one sample thenext, the system was cleaned with acetone using the steps described inTable 2. This wash procedure takes two minutes. TABLE 2 SequentialInjection Procedure for Cleaning Between Runs. Flow Volume, Rate,Procedural Step Solution Port μl μl/s 1 Place acetone vial at mix inlet2 Initialize mix inlet Acetone mix 3 Aspirate Acetone mix 90 50 4 Fillthe cell Acetone cell −85   200 5 Aspirate Acetone mix  450- 50 inletVol6 Remove acetone vial 7 Empty the inlet line Acetone mix inletVol 50 8Soak for 60 seconds 9 Discard through cell Acetone cell −450    50 10Rinse the mix inlet Wash mix -inletVol 50 11 Empty the inlet line Washmix inletVol 50 12 Rinse the cell Wash cell -inletVol- 200 150  13Restore the mix inlet

[0118] The automated acetone cleaning procedure outlined in Table 2 waseffective at removing TNT carryover from the semi-automated method up toand including the maximum tested concentration of 5000 ng/ml TNT. Thecarryover error without cleaning was characterized by running a sequenceof standards followed by blanks: 9.3, blank, 110, blank, 527, blank,5000 ng/ml, blank. Based on the resulting peak areas, the apparentconcentrations of the blanks were 0.1, 0.6, 1.2, and 1.1 ng/ml. Therewas no measurable carryover using the acetone cleaning procedure.

Example Three Methodology, Fully Automated Procedure

[0119] The automated method was the same as the semi-automated methodexcept that mixing of sample and reagents was automated by pullingsample, particle suspension, and TNT-HRP solution as stacked zones intothe holding coil. Zone volumes for sample, particles, and TNT-HRP were10 μl, 15 μl, and 15 μl, respectively, and the zone stacking wasrepeated 10 times to yield the total solution volumes of 100, 150, and150 μl. An important difference between the semi-automated and automatedprocedure is that in the automated procedure, free sample TNT is pulledinto the flow system and can adsorb to tubing or valve surfaces. In thesemi-automated procedure, the TNT has the opportunity to bind to themagnetic particles before being handled in the flow system.

[0120] The 3-part mixture was injected to the capture cell immediatelyafter zone stacking. Therefore, the competitive assay incubation time ofthe automated method was defined by the time required for zone stackingplus the time needed for capture of particles at the magnet, whichtotaled 4.25 minutes. The total procedure required 15.8 minutes,beginning with aspirating the solutions into the holding coil, andending with discarding the particles.

Calibration Stability Test—Automated Procedure

[0121] To evaluate the automated procedure and test the calibrationstability, 83 calibration standards from blank to 100 ng/ml were run bythe automated procedure over a time-span of 14 days. These automatedcalibration data are shown in FIG. 12.

[0122] The calibration was essentially stable for the two week testperiod, but carryover after some tests at the highest testconcentration, 100 ng/ml, was apparent in lower than expected resultsfor the next standard. These points are included in FIG. 12 with “X”markers on the plot, where they are particularly notable for blanks runafter the 100 ng/ml standard. These points were not included in thestatistics.

[0123] In tests without cleaning, 100 ng/mL caused a carryover effect ina subsequent blank, but lower concentrations did not. The automatedprocedure was run using DNA-zap, rather than acetone, as the cleaningsolution, and this did not prevent carryover problems at 100 ng/ml. (DNAZap™ is a two-part reagent that is harmless before mixing and afterreaction, which makes it very attractive as a flow system cleaningagent. It is a proprietary formulation for destroying DNA contaminationon surfaces prior to polymerase chain reaction amplification by cleavingof phosphate-sugar linkages.)

[0124] At the end of the two week calibration set, a 5000 ng/mL standardwas run and this caused persistent carryover that could not be removedwith either DNA-zap or acetone cleaning. Dismantling and cleaning thevalve was necessary to resolve this carryover. This stands in starkcontrast to the semi-automated method where 5000 ng/mL standards couldbe run routinely with acetone cleaning, and produced only 1.1 ng/mLapparent TNT concentration in a subsequent blank run without cleaning.

[0125] During method development, an aqueous mixture of 0.3 M KOH and0.03 M Na₂SO₃ was used at least 96 times in order to eliminate TNTcarryover by forming soluble anionic TNT adducts with hydroxide andsulfite. This method was generally satisfactory, but the acetoneprocedure that replaced it was more reliable.

Example Four Particle Capture and Resuspension Evaluation

[0126] Particle capture was evaluated by passing a 50 μl zone ofparticle suspension at various flow rates through the magnetic capturezone and measuring absorbance downstream. Relative to the peak area withno magnet present, the turbidity peak areas were 0.6% at 2.5 μl/s, 7% at5 μl/s, and 60% at 10 μl/s after correcting for the flow rate changes.Thus, 99% of the magnetic particles were captured at 2.5 μl/s. Therepeatability of particle capture and release is apparent in thecalibration stability results to be described below.

[0127] Sudden changes in flow rate from zero to 200 μl/s were used todisplace captured particles from the magnetic cell tubing wall. Both 200and 400 μl/s flow rates were found to be effective for particle release,as determined by having no carryover enzyme-activity in subsequent blankruns. However, 200 μls disperses the particles less than a 400 μl/spulse, and was preferred. Particle release was carried out in twodirections. After completion of the assay, particles were released totravel forward through the transmittance optical cell and discarded.During the procedure, particles were released with either a 5 or 15 μLpulse at 200 μL/s back toward the valve to resuspend them in the 22 μLzone between the magnet and the valve. This procedure was particularlyuseful for suspending the particles in the reagent solution for colordevelopment, but it was also used in the wash steps. After colordevelopment, the particles were recaptured in the capture zone as thesolution was pushed through the optical transmittance cell at 1 μL/s.Resuspending particles provides better surface accessibility for washand catalysis steps than leaving the particles in a layer against themagnetic cell wall. In addition, resuspension prevents particleaggregates from forming, which can be a problem if the magneticparticles are left in the magnetic zone too long. This is why particleswere resuspended with a 5 microliter pulse in steps 4 and 10 of Table 1,just before the air segment discard and color inlet initializationsteps. Air segments were used to separate the solutions pulled into theholding coil (competitive mix and color development reagent solution) asdescribed above.

Example Five Alternative Procedures

[0128] Two other approaches were considered. In one the unlabeledanalyte was added first, a sequential approach that is sometimes used toincrease sensitivity. Deshpande, S. S. Enzyme Immunoassays: From Conceptto Product Development; Chapman & Hall: New York, 1996, p. 309. When a4.265 ng/ml TNT standard was incubated for 16 minutes with antibodyparticles prior to addition of the TNT-HRP, the resulting peak area wasthe same as if the 3 reagents were mixed without the added incubation.The peak area predicted 4.3 ng/ml for this altered 4.265 ng/ml run.

[0129] In the converse experiment, particles were incubated withTNT-HRP, washed three times, and incubated with TNT. The colordevelopment portion of the assay was used as a measure of the boundTNT-HRP remaining after incubation. Displacement of TNT-HRP wassignificant only in the μg/ml range when TNT standard was incubated for16 minutes. A 5000 ng/ml TNT standard resulted in 71.7% of the blankpeak area (average of triplicate runs). In competitive assays, thisconcentration represented infinite TNT concentration, completelyeliminating HRP activity in the color development step.

[0130] An alternative experiment was conducted where atrazine-HRPconjugate (also from Strategic Diagnostics) was used in a blank run inplace of TNT-HRP, yielding a blank peak area 17% that of the TNT-HRParea resulted. These results indicate that the antibody-coated magneticparticles can also bind atrazine-HRP, suggesting that there may be someaffinity for HRP regardless of the conjugated analyte or analyte-analog.

[0131] All references, including publications, patents, and patentapplications, cited or listed in this specification are hereinincorporated by reference as if each individual reference werespecifically and individually indicated to be incorporated by referenceand set forth in its entirety herein. Further, any theory, proposedmechanism of operation, or finding stated herein is meant to furtherenhance understanding of the present invention, and is not intended toin any way limit the present invention to such theory, proposedmechanism of operation, or finding.

[0132] While the invention has been illustrated and described in detailin the drawings and foregoing description, the same is to be consideredas illustrative and not restrictive in character, it being understoodthat only the preferred embodiments have been shown and described andthat all changes and modifications that come within the spirit of theinvention are desired to be protected.

What is claimed is:
 1. A method comprising: providing a fluid flow path,the fluid flow path having first and second ends, a fluid flowcontroller effective to variably impose a positive or negative pressureon the flow path to cause controlled fluid flow through the fluid flowpath in a first or second direction at predetermined rates, and acapture zone between the first and second ends, wherein a fixed magneticfield intercepts the fluid flow path in the capture zone; providing inthe fluid flow path a first mixture including a plurality of solidmagnetic particles dispersed in a carrier medium; passing the firstmixture through the capture zone at a first predetermined capture ratewhereby a major portion of the magnetic particles become trapped in thecapture zone by the force of the magnetic field and thereby separatedfrom the carrier medium to form a first magnetic particle isolate;perfusing the first magnetic particle isolate with a first dispersionmedium; and pulsing the first dispersion medium through the capture zoneat a first predetermined dispersion rate effective to dislodge the firstmagnetic particle isolate from the capture zone, move the magneticparticles from the magnetic field, and suspend the magnetic particles inthe first dispersion medium to provide a second mixture.
 2. The methodin accordance with claim 1 wherein the fixed magnetic field has astrength that is substantially constant during said passing of the firstmixture, said perfusing, said pulsing and said passing of the secondmixture.
 3. The method in accordance with claim 1 wherein the fixedmagnetic field is provided by a permanent magnet having a fixedrelationship to the fluid flow path.
 4. The method in accordance withclaim 1 wherein the fixed magnetic field is provided by an electromagnetthat has a fixed relationship to the fluid flow path and a that produceda fixed magnetic field.
 5. The method in accordance with claim 1 whereinthe magnetic particles include a surface-bound selective agent featuringselective affinity for a target substance.
 6. The method in accordancewith claim 5 wherein the magnetic particles with surface-bound selectiveagents are effective to selectively retain a chemical or biologicalspecies in a sample.
 7. The method in accordance with claim 1 whereinthe magnetic particles include properties suitable for adsorption ofmultiple target substances via a non-specific interaction.
 8. The methodin accordance with claim 7 wherein the non-specific interaction isselected from the group consisting of an electrostatic interaction, avan der Waals interaction, dipole-dipole interaction and a hydrogenbonding interaction.
 9. The method in accordance with claim 6 whereinthe first dispersion medium includes the sample.
 10. The method inaccordance with claim 6 wherein the selective agent is effective toretain a biological species in a sample, and wherein the selective agentis selected from the group consisting of an antigen, an antibody, aprotein receptor, a ligand, an oligonucleotide, streptavidin, avidin,biotin and lectin.
 11. The method in accordance with claim 10 whereinthe selective agent is an antibody.
 12. The method in accordance withclaim 6, wherein the sample includes an analyte sample for an ELISAassay; wherein the selective agent is an antibody that selectivelyretains the analyte; and wherein the sample further includes ananalyte-enzyme conjugate.
 13. The method in accordance with claim 12,wherein the analyte-enzyme conjugate comprises an enzyme having aspecific catalytic activity and specificity for an analysis reagent. 14.The method in accordance with claim 6 wherein the carrier mediumincludes the sample.
 15. The method in accordance with claim 14 whereinthe mixture is incubated for a first period of time effective to causethe selective agent to contact and retain the species.
 16. The method inaccordance with claim 15 wherein the first dispersion medium is a firstwash solution.
 17. The method in accordance with claim 16, furthercomprising passing the second mixture through the capture zone at asecond predetermined capture rate at which rate a major portion of themagnetic particles become trapped in the capture zone by the force ofthe magnetic field and thereby removed from the first dispersion mediumto form a second magnetic particle isolate.
 18. The method in accordancewith claim 17, further comprising: perfusing the second magneticparticle isolate with a second dispersion medium; and pulsing the seconddispersion medium through the capture zone at a second predetermineddispersion rate effective to dislodge the second magnetic particleisolate from the capture zone, move the magnetic particles from themagnetic field, and suspend the magnetic particles in the seconddispersion medium to provide a third mixture.
 19. The method inaccordance with claim 18 wherein the second dispersion medium comprisesan analysis reagent.
 20. The method in accordance with claim 19, furthercomprising passing the third mixture through the capture zone at a thirdpredetermined capture rate at which rate a major portion of the magneticparticles become trapped in the capture zone by the force of themagnetic field and thereby removed from the second dispersion medium toform a third magnetic particle isolate.
 21. The method in accordancewith claim 20 wherein the flow path further includes a detection zone,and wherein a detector is positioned to detect a physical or chemicalproperty of a fluid in the detection zone; and further comprising, afterpassing the third mixture through the capture zone, detecting a physicalor chemical property of a member selected from the group consisting ofthe second dispersion medium and the third magnetic particle isolate.22. The method in accordance with claim 21 wherein the analysis reagentis a coloring agent; and wherein the detector is an optical detector.23. The method in accordance with claim 22, further comprising: passingthe second solution through the detection zone; and measuring theproperty of the second solution.
 24. The method in accordance with claim18 wherein the second dispersion medium comprises a second washsolution.
 25. The method in accordance with claim 24, further comprisingpassing the third mixture through the capture zone at a thirdpredetermined capture rate at which rate a major portion of the magneticparticles become trapped in the capture zone by the force of themagnetic field and thereby removed from the second dispersion medium toform a third magnetic particle isolate.
 26. The method in accordancewith claim 25, further comprising: perfusing the third magnetic particleisolate with a third dispersion medium; and pulsing the third dispersionmedium through the capture zone at a third predetermined dispersion rateeffective to dislodge the third magnetic particle isolate from thecapture zone, move the magnetic particles from the magnetic field, andsuspend the magnetic particles in the third dispersion medium to providea fourth mixture.
 27. The method in accordance with claim 26, furthercomprising passing the fourth mixture through the capture zone at afourth predetermined capture rate at which rate a major portion of themagnetic particles become trapped in the capture zone by the force ofthe magnetic field and thereby removed from the third dispersion mediumto form a fourth magnetic particle isolate.
 28. The method in accordancewith claim 27 wherein the flow path further includes a detection zone,and wherein a detector is positioned to detect a physical or chemicalproperty of a fluid in the detection zone; and further comprising, afterpassing the fourth mixture through the capture zone, detecting aphysical or chemical property of a member selected from the groupconsisting of the third dispersion medium and the fourth magneticparticle isolate.
 29. The method in accordance with claim 28 wherein thesecond dispersion medium comprises an analysis reagent.
 30. The methodin accordance with claim 29 wherein the analysis reagent is a coloringagent; and wherein the detector is an optical detector.
 31. The methodin accordance with claim 1 wherein the magnetic field has a fieldgradient in the capture zone of from about 0.1 to about 2 kGauss/cm. 32.The method in accordance with claim 1 wherein the flow path has a volumeof from about 0.01 to about 50 μL.
 33. The method in accordance withclaim 1 wherein the flow path has an average diameter of from about0.001 to about 5 mm.
 34. The method in accordance with claim 1 whereinthe first predetermined capture rate is from about 1.0 to about 13 mm/s.35. The method in accordance with claim 1 wherein the firstpredetermined dispersion rate is from about 250 to about 2500 mm/s. 36.The method in accordance with claim 1 wherein the flow path is amicrochannel.
 37. The method in accordance with claim 1 wherein saidpassing of the first mixture comprises passing in a first direction;wherein said passing of the first dispersion medium comprises passing ina second direction opposite the first direction; and wherein saidpassing of the second mixture comprises passing in the first direction.38. The method in accordance with claim 1 wherein the flow controllercomprises: a multiport selection valve including a primary port and aplurality of secondary ports, wherein a first secondary port is fluidlyconnected to the inlet of the fluid flow path; a holding coil having aproximal end and a distal end; wherein the distal end is fluidlyconnected to the primary port of the selection valve; a three-way valvehaving a first port fluidly connected to the proximal end of the holdingcoil; a second port fluidly connected to a variable speed reversiblepump; and a third port fluidly connected to a source of a washcomposition.
 39. The method in accordance with claim 38 wherein thevariable speed reversible pump is a stepper-motor-driven syringe pump.40. The method in accordance with claim 38 wherein the multiportselection valve, the three-way valve and the pump are controlled by apre-programmed computer.
 41. The method in accordance with claim 1,wherein the flow path in the capture zone is substantially free fromfixed magnetizable solid matrix structures.
 42. An apparatus,comprising: a fluid flow path, the fluid flow path having first andsecond ends and a capture zone between the first and second ends; afluid flow controller effective to variably impose a positive ornegative pressure on the flow path to cause controlled fluid flowthrough the fluid flow path in a first or second direction atpredetermined rates; a magnetic field source generating a fixed magneticfield, the source positioned in a fixed relationship to the fluid flowpath whereby the field intercepts the fluid flow path in the capturezone; and a detector positioned to detect a physical or chemicalproperty of a fluid in the flow path.
 43. The apparatus in accordancewith claim 42 wherein the detector is an optical detector.
 44. Theapparatus in accordance with claim 42 wherein the fixed magnetic fieldsource comprises a permanent magnet.
 45. The apparatus in accordancewith claim 42 wherein the fixed magnetic field source comprises anelectromagnet.
 46. The apparatus in accordance with claim 42 wherein themagnetic field has a field strength of from about 0.1 to about 2kGauss/cm.
 47. The apparatus in accordance with claim 42 wherein theflow path in the capture/dispersion region has a volume of from about 1to about 50 μL.
 48. The apparatus in accordance with claim 42 whereinthe flow path has an average diameter of from about 0.1 to about 5 mm.49. The apparatus in accordance with claim 42 wherein the fluid flowcontroller is effective to provide flow rates in the fluid flow path ofup to about 2500 mm/s in either direction.
 50. The apparatus inaccordance with claim 42 wherein the controller includes a reversiblepump in fluid communication with the fluid flow path.
 51. The apparatusin accordance with claim 42 wherein the fluid flow controller comprises:a multiport selection valve including a primary port and a plurality ofsecondary ports, wherein a first secondary port is fluidly connected tothe inlet of the fluid flow path; a holding coil having a proximal endand a distal end; wherein the distal end is fluidly connected to themain port of the selection valve; a three-way valve having a first portfluidly connected to the proximal end of the holding coil; a second portfluidly connected to a variable speed reversible pump; and a third portfluidly connected to a source of a wash composition.
 52. The apparatusin accordance with claim 42 wherein the variable speed reversible pumpis a stepper-motor-driven syringe pump.
 53. The apparatus in accordancewith claim 42 wherein the multiport selection valve, the three-way valveand the pump are controlled by a pre-programmed computer.
 54. Theapparatus in accordance with claim 42 wherein the flow path in thecapture zone is substantially free from fixed magnetizable solid matrixstructures.
 55. An apparatus, comprising: a fluid flow path, the fluidflow path having first and second ends and a plurality of capture zonesbetween the first and second ends; a fluid flow controller effective tovariably impose a positive or negative pressure on the flow path tocause controlled fluid flow through the fluid flow path in a first orsecond direction at predetermined rates; and a plurality of magneticfield sources, each source generating a fixed magnetic field, and eachthe source positioned in a fixed relationship to the fluid flow pathwhereby each field intercepts the fluid flow path in a capture zone;wherein each capture zone is separated from one or more other capturezones by a zone that is substantially free from a magnetic field.
 56. Anapparatus, comprising: a fluid flow path means having first and secondends and a capture zone between the first and second ends; means forproviding in the fluid flow path a first mixture including a pluralityof solid magnetic particles dispersed in a carrier medium; means forvariably imposing a positive or negative pressure on the fluid flow pathmeans to cause controlled fluid flow through the fluid flow path in afirst or second direction at predetermined rates; and means forproviding a fixed magnetic field that intercepts the fluid flow pathmeans in the capture zone.
 57. A method comprising: passing a mixtureincluding a plurality of solid magnetic particles dispersed in a carriermedium through a conduit extending through a fixed magnetic field at afirst predetermined capture rate whereby magnetic particles becometrapped in the field and thereby separated from the carrier medium toform a magnetic particle isolate; passing a dispersion medium into theconduit and into contact with the magnetic particle isolate; and pulsingthe dispersion medium through the conduit at a first predetermineddispersion rate effective to move the magnetic particles from themagnetic field and suspend the magnetic particles in the dispersionmedium.
 58. A method comprising: passing a first mixture including aplurality of solid magnetic particles dispersed in a carrier mediumthrough a conduit extending through a magnetic field at a firstpredetermined capture rate whereby magnetic particles become trapped inthe field and thereby separated from the carrier medium to form amagnetic particle isolate; releasing the magnetic particle isolate fromthe magnetic field by pulsing a dispersion medium through the conduit ata first predetermined dispersion rate effective to move the magneticparticles from the magnetic field and suspend the magnetic particles inthe dispersion medium to provide a second mixture; and recapturing themagnetic particles by passing the second mixture through the magneticfield in a direction opposite said passing of the first mixture, at asecond predetermined capture rate whereby magnetic particles becometrapped in the field and thereby separated from the carrier medium toform a magnetic particle isolate.
 59. The method in accordance withclaim 58 wherein the first mixture has a different composition than thesecond mixture.